title: Ubiquitin binding modulates IAP antagonist-stimulated proteasomal degradation of c-IAP1 and c-IAP2(1).
abstract: A family of anti-apoptotic regulators known as IAP (inhibitor of apoptosis) proteins interact with multiple cellular partners and inhibit apoptosis induced by a variety of stimuli. c-IAP (cellular IAP) 1 and 2 are recruited to TNFR1 (tumour necrosis factor receptor 1)-associated signalling complexes, where they mediate receptor-induced NF-kappaB (nuclear factor kappaB) activation. Additionally, through their E3 ubiquitin ligase activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NIK (NF-kappaB-inducing kinase) and regulate the non-canonical NF-kappaB pathway. In the present paper, we describe a novel ubiquitin-binding domain of IAPs. The UBA (ubiquitin-associated) domain of IAPs is located between the BIR (baculovirus IAP repeat) domains and the CARD (caspase activation and recruitment domain) or the RING (really interesting new gene) domain of c-IAP1 and c-IAP2 or XIAP (X-linked IAP) respectively. The c-IAP1 UBA domain binds mono-ubiquitin and Lys(48)- and Lys(63)-linked polyubiquitin chains with low-micromolar affinities as determined by surface plasmon resonance or isothermal titration calorimetry. NMR analysis of the c-IAP1 UBA domain-ubiquitin interaction reveals that this UBA domain binds the classical hydrophobic patch surrounding Ile(44) of ubiquitin. Mutations of critical amino acid residues in the highly conserved MGF (Met-Gly-Phe) binding loop of the UBA domain completely abrogate ubiquitin binding. These mutations in the UBA domain do not overtly affect the ubiquitin ligase activity of c-IAP1 or the participation of c-IAP1 and c-IAP2 in the TNFR1 signalling complex. Treatment of cells with IAP antagonists leads to proteasomal degradation of c-IAP1 and c-IAP2. Deletion or mutation of the UBA domain decreases this degradation, probably by diminishing the interaction of the c-IAPs with the proteasome. These results suggest that ubiquitin binding may be an important mechanism for rapid turnover of auto-ubiquitinated c-IAP1 and c-IAP2.
authors.affiliation: Department of Protein Engineering, Genentech, Inc., 1 DNA Way, M/S 40, South San Francisco, CA 94080, USA.
authors.forename: John W
authors.initials: JW
authors.lastname: Blankenship
authors.forename: Eugene
authors.initials: E
authors.lastname: Varfolomeev
authors.forename: Tatiana
authors.initials: T
authors.lastname: Goncharov
authors.forename: Anna V
authors.initials: AV
authors.lastname: Fedorova
authors.forename: Donald S
authors.initials: DS
authors.lastname: Kirkpatrick
authors.forename: Anita
authors.initials: A
authors.lastname: Izrael-Tomasevic
authors.forename: Lilian
authors.initials: L
authors.lastname: Phu
authors.forename: David
authors.initials: D
authors.lastname: Arnott
authors.forename: Mariam
authors.initials: M
authors.lastname: Aghajan
authors.forename: Kerry
authors.initials: K
authors.lastname: Zobel
authors.forename: J Fernando
authors.initials: JF
authors.lastname: Bazan
authors.forename: Wayne J
authors.initials: WJ
authors.lastname: Fairbrother
authors.forename: Kurt
authors.initials: K
authors.lastname: Deshayes
authors.forename: Domagoj
authors.initials: D
authors.lastname: Vucic
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002151:Calorimetry; D002460:Cell Line; D045744:Cell Line, Tumor; D002942:Circular Dichroism; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D025821:Polyubiquitin / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination; D000097782:NF-kappaB-Inducing Kinase
chemical_list: D051034:Inhibitor of Apoptosis Proteins; D016328:NF-kappa B; D025821:Polyubiquitin; D046988:Proteasome Endopeptidase Complex; D017346:Protein Serine-Threonine Kinases; D047888:Receptors, Tumor Necrosis Factor, Type I; D025801:Ubiquitin; D000097782:NF-kappaB-Inducing Kinase